Dual interactions of the translational repressor Paip2 with poly(A) binding protein - PubMed (original) (raw)

Dual interactions of the translational repressor Paip2 with poly(A) binding protein

K Khaleghpour et al. Mol Cell Biol. 2001 Aug.

Abstract

The cap structure and the poly(A) tail of eukaryotic mRNAs act synergistically to enhance translation. This effect is mediated by a direct interaction of eukaryotic initiation factor 4G and poly(A) binding protein (PABP), which brings about circularization of the mRNA. Of the two recently identified PABP-interacting proteins, one, Paip1, stimulates translation, and the other, Paip2, which competes with Paip1 for binding to PABP, represses translation. Here we studied the Paip2-PABP interaction. Biacore data and far-Western analysis revealed that Paip2 contains two binding sites for PABP, one encompassing a 16-amino-acid stretch located in the C terminus and a second encompassing a larger central region. PABP also contains two binding regions for Paip2, one located in the RNA recognition motif (RRM) region and the other in the carboxy-terminal region. A two-to-one stoichiometry for binding of Paip2 to PABP with two independent K(d)s of 0.66 and 74 nM was determined. Thus, our data demonstrate that PABP and Paip2 could form a trimeric complex containing one PABP molecule and two Paip2 molecules. Significantly, only the central Paip2 fragment, which binds with high affinity to the PABP RRM region, inhibits PABP binding to poly(A) RNA and translation.

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Figures

FIG. 1

Identification of PABP binding sites in Paip2. Purified Paip2 and Paip2 fragments (0.1 μg) were resolved by SDS–15% polyacrylamide gel electrophoresis and electroblotted onto a nitrocellulose membrane. The membrane was probed with a rabbit polyclonal anti-GST antibody (A) or processed for far-Western analysis using 32P-labeled FLAG-HMK-PABP as a probe (B), as described in Materials and Methods. Positions of molecular weight markers are shown at right. (C) Schematic diagram of the GST-Paip2 fragments with a summary of the PABP-Paip2 interaction results. Black boxes represent the region rich in glutamic acids, and grey represents the Paip2 C-terminal PABP binding site. The sequences at the top exhibit homology to Paip1.

FIG. 1

FIG. 2

Identification of Paip2 binding sites in PABP. PABP fragments were resolved by SDS–15% polyacrylamide gel electrophoresis and electroblotted onto a nitrocellulose membrane. The blots were probed with a rabbit polyclonal anti-GST antibody or processed for far-Western analysis using 32P-labeled HMK-Paip2 as a probe, as described in Materials and Methods. Lanes contain the following amounts of protein. (A) Individual RRMs, 1 μg each; PABP-C1 and -C2, 0.5 μg; GST, 1 μg; and PABP-His, 0.5 μg. (B) Combinations of RRMs, 0.5 μg each; PABP-C2, 0.5 μg; GST, 1 μg; and PABP-His, 0.5 μg. Positions of molecular weight markers are shown at right. (C) Schematic diagram of the GST-PABP fragments with a summary of the Paip2-PABP interaction results. Shaded areas represent domains that contain Paip2 interaction sites in PABP.

FIG. 3

SPR analysis of the interaction between PABP and Paip2. Paip2 (1, 4, 16, and 64 nM; cyan, black, blue and red lines, respectively) was injected over a PABP surface (800 RU) and over a mock surface (no PABP coupled). Data were treated and integrated using a simple model (A) or models depicting a rearrangement of the protein complex (B) or the existence of two independent binding sites in PABP (C), as described in Materials and Methods. Top panels: experimental sensorgrams (dots) and the calculated fits (solid lines). Bottom panels: corresponding residuals (difference between calculated and experimental data points). Kinetic constants are listed in Table 1.

FIG. 4

SPR analysis of the interaction between PABP RRM1-4 or RRM2-3 and Paip2. RRM1-4 (3.12, 6.25, 25, and 100 nM; cyan, red, blue and black lines, respectively) (A) or RRM2-3 (3.12, 6.25, 25, and 100 nM; green, red, blue and black lines, respectively) (B) were injected over a Paip2 surface (250 RU) and over a mock surface. Data were treated and integrated with a simple model. Top panels: experimental sensorgrams (dots) and the calculated fits (solid lines). Bottom panels: corresponding residuals. Kinetic constants are listed in Table 2.

FIG. 5

SPR analysis of the interaction between PABP-C2 and Paip2. Paip2 (1, 4, 16, and 64 nM; black, blue, red and cyan lines, respectively) was injected over a PABP-C2 surface (1,800 RU) and over a mock surface. Data were treated and integrated with a simple model (A) or with models depicting a conformational change (rearrangement) of the protein complex (B) or the existence of two independent binding sites in PABP-C2 (C). Top panels: experimental sensorgrams (dots) and the calculated fits (solid lines). Bottom panels: corresponding residuals. Kinetic constants are listed in Table 3.

FIG. 6

Binding of recombinant PABP fragments to Paip2 fragments. GST pull-down of PABP RRMs 1 to 4 (A), PABP RRMs 2 and 3 (B), PABP-C2 (C), or no PABP with GST-Paip2 fragments (D). Proteins (2 μg) were incubated with glutathione 4B-Sepharose (25 μl) for 1 h at 4°C and washed four times with 1 ml of buffer A. Bound proteins were eluted by boiling samples in 2× Laemmli sample buffer and resolved by SDS–12.5% or 15 to 20% polyacrylamide gel electrophoresis. The gel was stained with Coomassie R-250. Positions of molecular weight markers are shown at right.

FIG. 7

Functional dissection of Paip2. (A) Coomassie R-250 staining of wild-type (wt) and indicated truncated mutants of GST-Paip2. The positions of prestained molecular weight markers are also shown. (B) Effects of Paip2 mutants on translation. Krebs-2 cell-free translation reactions (12.5 μl) were programmed with 25 ng of capped poly(A)+ luciferase mRNA in the absence or presence of GST-Paip2 wild type or the indicated GST-Paip2 mutants at 30°C for 60 min, as described previously (21). Following incubation, 3-μl aliquots were assayed for luciferase activity using the luciferase assay kit (Promega) in a Lumat LB 9507 bioluminometer (EG&G Berthold). Relative luciferase activities (average of two independent determinations) are shown; the value obtained in the absence of added GST-Paip2 was set as 100%. (C) Inhibition of PABP binding to poly(A) by Paip2 mutants. Filter binding assays were performed as described in Materials and Methods. His-PABP (10 nM) and various GST-Paip2 fusion proteins (10 or 100 nM) were incubated with 32P-labeled A25 RNA. Reaction mixtures were then filtered through a nitrocellulose membrane. The radioactivity corresponding to the A25 RNA, which was retained on the membrane in the presence of PABP alone, was set at 100%. The relative levels of retention of the A25 RNA for the different GST-Paip2 proteins are shown. Each result shown is the average of results of at least two independent experiments, which did not differ by more than 10%. (D) Effect of wild-type and mutant GST-Paip2 on the poly(A)-organizing activity of PABP. The poly(A)-organizing activity of PABP was assayed in a total volume of 50 μl with radiolabeled poly(A) (0.5 × 106 cpm) and His-PABP (0.15 μg, 2.1 pmol) essentially as described previously (3, 21). GST-Paip2 wild-type or mutant proteins either were not added (lanes 1, 5, 9, 13, and 17) or were present in the reactions at 2- (lanes 2, 6, 10, 14, and 18), 5- (lanes 3, 7, 11, 15, and 19), and 10-pmol (lanes 4, 8, 12, 16, and 20) amounts. Following PABP-poly(A) complex formation, the mixtures were subjected to limited digestion with micrococcal nuclease and analyzed on a 7 M urea-containing 10% polyacrylamide gel (21).

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Dual interactions of the translational repressor Paip2 with poly(A) binding protein - PubMed (original) (raw)